Mutant of propionyl-CoA transferase from Clostridium propionicum and preparing method for PLA or PLA copolymer using the same

ABSTRACT

Provided is a mutant of propionyl-CoA transferase from  Clostridium propionicum  that can convert lactate into lactyl-CoA with high efficiency in a method of preparing a polylactate (PLA) or PLA copolymer using microorganisms. Unlike conventional propionyl-CoA transferase which is weakly expressed in  E. coli , when a mutant of propiony-CoA transferase from  Clostridium propionicum  is introduced into recombinant  E. coli , lactyl-CoA can be supplied very smoothly, thereby enabling highly efficient preparation of polylactate (PLA) and PLA copolymer.

This application is a Divisional of U.S. patent application Ser. No.12/673,389, filed on Feb. 12, 2010, which is a National StageApplication of PCT/KR2008/004348, filed on Jul. 25, 2008 and claims thebenefit of Korean Patent Application No. 10-2007-0081855, filed Aug. 14,2007, the disclosures of which are incorporated by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to a mutant of propionyl-CoA transferasefrom Clostridium propionicum, which can convert lactate into lactyl-CoAwith high efficiency in a method of preparing a polylactate (PLA) or PLAcopolymer using microorganisms.

BACKGROUND ART

Polylactate (PLA) is a typical biodegradable polymer derived fromlactate that is highly applicable commercially and biomedically.Although preparation of PLA presently involves polymerization of lactateproduced by fermenting microorganisms, only PLA with a low molecularweight of about 1000 to 5000 daltons is obtained by directpolymerization of lactate. In order to synthesize PLA with a molecularweight of 100,000 daltons or higher, PLA with a low molecular weightobtained by direct polymerization of lactate may be polymerized using achain coupling agent. In this method, however, the entire processbecomes complicated due to addition of an organic solvent or a chaincoupling agent, which is not easy to remove. A presently commerciallyavailable process of preparing high-molecular weight PLA may includeconverting lactate into lactide and synthesizing PLA using ring-openingpolycondensation of lactide rings.

When PLA is synthesized by chemical synthesis of lactate, a PLAhomopolymer is easily obtained, but a PLA copolymer composed of varioustypes of monomers is difficult to synthesize and commerciallyunavailable.

Meanwhile, polyhydroxyalkanoate (PHA) is polyester stored bymicroorganisms as energy or a carbon source when there are excessivecarbon sources and a lack of other nutrients, such as phosphorus (P),nitrogen (N), magnesium (Mg) and oxygen (O), etc. Since PHA has similarphysical properties to a conventional synthetic polymer from petroleumand exhibits complete biodegradability, it is being recognized as asubstitute for conventional synthetic plastics.

In order to produce PHA using microorganisms, an enzyme for convertingmicrobial metabolic products into a PHA monomer, and PHA synthase forsynthesizing a PHA polymer using the PHA monomer, are needed. Whensynthesizing PLA and PLA copolymer using microorganisms, the same systemis required, and an enzyme for providing lactyl-CoA is needed inaddition to an enzyme for providing hydroxyacyl-CoA, which is anoriginal substrate of PHA synthase.

Therefore, in order to provide lactyl-CoA, the present inventors usedpropionyl-CoA transferase from Clostridium propionicum and a mutant ofPHA synthase from Pseudomonas sp. 6-19 using the propionyl-CoAtransferase as a substrate, thus could successfully synthesize PLA andPLA copolymer as disclosed in Korean Patent Application NO.10-2006-0116234.

However, it was reported that when propionyl-CoA transferase fromClostridium propionicum, which is an enzyme for supplying lactyl-CoA, ishighly expressed in E. coli by a very potent promoter, serious metabolicdisorder occurs, thus inhibiting cell growth (Selmer et al. reported inEur. J. Biochem. 269:372, 2002). Also, since the codon usage of a genefor encoding propionyl-CoA transferase from Clostridium propionicum isquite different from that of E. coli, it may be very difficult tonormally express propionyl-CoA transferase. Accordingly, in order tosynthesize PLA and PLA copolymer more efficiently than conventionalsystems, it is very important to introduce propionyl-CoA transferase,which smoothly provides lactyl-CoA and is expressed enough not togreatly inhibit cell growth.

DISCLOSURE Technical Problem

The present invention is directed to a method of preparing a polylactate(PLA) or PLA copolymer with high efficiency by providing amonomer-supplying enzyme capable of efficiently supplying lactyl-CoA.

Technical Solution

The present inventors found that when a mutant of propionyl-CoAtransferase from Clostridium propionicum is used, a PLA or PLA copolymercan be prepared with high efficiency by efficiently supplying lactyl-CoAwithout greatly inhibiting cell growth. This led them to complete thepresent invention.

Accordingly, the present invention provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3, in which T78C, T669C, A1125G and T1158Care mutated, and a gene encoding the mutant.

The present invention also provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3 in which A1200G is mutated, and a geneencoding the mutant.

The present invention also provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3 in which A1200G is mutated and an aminoacid sequence corresponding to SEQ ID NO: 3 in which Gly335Asp ismutated, and a gene encoding the mutant.

The present invention also provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3 in which T669C, A1125G and T1158C aremutated, and an amino acid sequence corresponding to SEQ ID NO: 3 inwhich Asp65Gly is mutated, and a gene encoding the mutant.

The present invention also provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3 in which T669C, A1125G and T1158C aremutated and an amino acid sequence corresponding to SEQ ID NO: 3 inwhich Asp65Asn is mutated, and a gene encoding the mutant.

The present invention also provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3 in which T669C, A1125G and T1158C aremutated and an amino acid sequence corresponding to SEQ ID NO: 3 inwhich Thr1991Ile is mutated, and a gene encoding the mutant.

The present invention also provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3 in which A1200G is mutated and an aminoacid sequence corresponding to SEQ ID NO: 3 in which Ala243Thr ismutated, and a gene encoding the mutant.

The present invention also provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3 in which A1200G is mutated and an aminoacid sequence corresponding to SEQ ID NO: 3 in which Asp257Asn ismutated, and a gene encoding the mutant.

The present invention also provides a mutant of propionyl-CoAtransferase functioning as a lactyl-CoA-supplying enzyme, which has agene sequence of SEQ ID NO: 3 in which T78C, T669C, A1125G and T1158Care mutated and an amino acid sequence corresponding to SEQ ID NO: 3 inwhich Val193Ala is mutated, and a gene encoding the mutant.

The present invention also provides a recombinant vector forsynthesizing a polylactate (PLA) or PLA copolymer, which contains anyone of the above-described genes.

Preferably, the recombinant vector for synthesizing a polylactate (PLA)or PLA copolymer contains any one of the above-described genes encodinga mutant of propionyl-CoA transferase and a polyhydroxyalkanoate (PHA)synthase gene (phaC1_(Ps6-19)300) of SEQ ID NO: 4, which is capable ofsynthesizing a PLA or PLA copolymer using lactyl-CoA as a substrate.

The present invention also provides a cell or plant transformed with oneof the above-described recombinant vectors. In this case, a cell orplant obtained by transforming a cell or plant without a gene ofpropionyl-CoA transferase with one of the above-described recombinantvectors is also within the scope of the present invention.

The present invention also provides a method of preparing a polylactate(PLA) or PLA copolymer, comprising culturing or growing theabove-described cell or plant is cultured or grown. In order to preparea PLA copolymer, culturing or growing the cell or plant may be performedin an environment containing hydroxyalkanoate. The hydroxyalkanoate maybe at least one selected from the group consisting of 3-hydroxybutyrate,3-hydroxyvalerate, 4-hydroxybutyrate, medium chain-length(D)-3-hydroxycarboxylic acid with 6 to 14 carbon atoms,2-hydroxypropionic acid, 3-hydroxypropionic acid, 3-hydroxyhexanoicacid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoicacid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid,3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid,3-hydroxyhexadecanoic acid, 4-hydroxyvaleric acid, 4-hydroxyhexanoicacid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxydecanoicacid, 5-hydroxyvaleric acid, 5-hydroxyhexanoic acid, 6-hydroxydodecanoicacid, 3-hydroxy-4-pentenoic acid, 3-hydroxy-4-trans-hexenoic acid,3-hydroxy-4-cis-hexenoic acid, 3-hydroxy-5-hexenoic acid,3-hydroxy-6-trans-octenoic acid, 3-hydroxy-6-cis-octenoic acid,3-hydroxy-7-octenoic acid, 3-hydroxy-8-nonenoic acid,3-hydroxy-9-decenoic acid, 3-hydroxy-5-cis-dodecenoic acid,3-hydroxy-6-cis-dodecenoic acid, 3-hydroxy-5-cis-tetradecenoic acid,3-hydroxy-7-cis-tetradecenoic acid, 3-hydroxy-5,8-cis-cis-tetradecenoicacid, 3-hydroxy-4-methylvaleric acid, 3-hydroxy-4-methylhexanoic acid,3-hydroxy-5-methylhexanoic acid, 3-hydroxy-6-methylheptanoic acid,3-hydroxy-4-methyloctanoic acid, 3-hydroxy-5-methyloctanoic acid,3-hydroxy-6-methyloctanoic acid, 3-hydroxy-7-methyloctanoic acid,3-hydroxy-6-methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid,3-hydroxy-8-methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid,3-hydroxy-9-methyldecanoic acid, 3-hydroxy-7-methyl-6-octenoic acid,malic acid, 3-hydroxysuccinic acid-methylester, 3-hydroxyadipinicacid-methylester, 3-hydroxysuberic acid-methyl ester, 3-hydroxyazelaicacid-methyl ester, 3-hydroxysebacic acid-methylester, 3-hydroxysubericacid-ethylester, 3-hydroxysebacic acid-ethylester, 3-hydroxypimelicacid-propylester, 3-hydroxysebacic acid-benzylester,3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid,phenoxy-3-hydroxybutyric acid, phenoxy-3-hydroxyvaleric acid,phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid,para-cyanophenoxy-3-hydroxybutyric acid,para-cyanophenoxy-3-hydroxyvaleric acid,para-cyanophenoxy-3-hydroxyhexanoic acid,para-nitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5-phenylvalericacid, 3-hydroxy-5-cyclohexylbutyric acid, 3,12-dihydroxydodecanoic acid,3,8-dihydroxy-5-cis-tetradecenoic acid, 3-hydroxy-4,5-epoxydecanoicacid, 3-hydroxy-6,7-epoxydodccanoic acid,3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid,7-cyano-3-hydroxyheptanoic acid, 9-cyano-3-hydroxynonanoic acid,3-hydroxy-7-fluoroheptanoic acid, 3-hydroxy-9-fluorononanoic acid,3-hydroxy-6-chlorohexanoic acid, 3-hydroxy-8-chlorooctanoic acid,3-hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid,3-hydroxy-11-bromoundecanoic acid, 3-hydroxy-2-butenoic acid,6-hydroxy-3-dodecenoic acid, 3-hydroxy-2-methylbutyric acid,3-hydroxy-2-methylvaleric acid, and 3-hydroxy-2,6-dimethyl-5-heptenoicacid. However, the present invention is not limited thereto.

The hydroxyalkanoate may be at least one selected from the groupconsisting of 3-hydroxybutyrate, 4-hydroxybutyrate, 2-hydroxypropionicacid, 3-hydroxypropionic acid, medium chain-length(D)-3-hydroxycarboxylic acid with 6 to 14 carbon atoms,3-hydroxyvalerate, 4-hydroxyvaleric acid, and 5-hydroxyvaleric acid.More preferably, but not necessarily, the hydroxyalkanoate may be3-hydroxybutyrate (3-HB) (refer to FIG. 1).

Accordingly, the PLA or PLA copolymer of the present invention may bepolylactate, poly(hydroxyalkanoate-co-3-lactate),poly(hydroxyalkanoate-co-hydroxyalkanoate-co-lactate),poly(hydroxyalkanoate-co-hydroxyalkanoate-co-polyhydroxyalkanoate-co-lactate)and so on, but the present invention is not limited thereto.

For example, the PLA copolymer may bepoly(4-hydroxybutyrate-co-lactate),poly(4-hydroxybutyrate-co-3-hydroxypropionate-co-lactate),poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-lactate),poly(3-hydroxybutyrate-co-3-hydroxypropionate-co-4-hydroxybutyrate-co-lactate),poly(medium chain length (MCL) 3-hydroxyalkanoate-co-lactate),poly(3-hydroxybutyrate-co-MCL 3-hydroxyalkanoate-co-lactate),poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-lactate),poly(3-hydroxybutyrate-co-3-hydroxypropionate-co-lactate),poly(3-hydroxypropionate-co-lactate) and so on, but the presentinvention is not limited thereto.

In the present invention, a vector refers to a DNA construct containinga DNA sequence that is operably linked to a suitable control sequencecapable of expressing DNA in an appropriate host. The vector may be aplasmid, a phage particle, or a simply latent genome insert. When thevector is transformed into an appropriate host, the vector is capable ofreplicating or functioning irrespective of a host genome or, in somecases, being integrated with the genome itself. Since a plasmid ispresently the most general type of vectors, the plasmid and vector maysometimes be used interchangeably in the present invention. However, thepresent invention includes other types of vectors known to one skilledin the art or those with equivalent functions.

The term “expression control sequence” means a DNA sequence essentialfor expression of an operably linked coding sequence in a specific host.The control sequence includes a promoter required for transcription, anarbitrary operator sequence for controlling the transcription, asequence for coding an appropriate mRNA ribosome binding site (RBS), anda sequence for controlling termination of transcription and translation.For example, a control sequence suitable for a prokaryote includes apromoter, a random operator sequence, and an RBS. A control sequencesuitable for a eukaryotic cell includes a promoter, a polyadenylationsignal, and an enhancer. The most important factor that affects theexpression amount of a gene in a plasmid is a promoter. A SRα promoteror a cytomegalovirus promoter may be used as a high-expression promoter.

In order to express a DNA sequence of the present invention, any one ofvarious expression control sequences may be used for a vector. Theexpression control sequence may be, for example, early and latepromoters of SV40 or adenovirus, the lac system, the trp system, the tacsystem, the trc system, T3 and T7 promoters, the major operator andpromoter regions of phage λ, the control region of fd code protein, thepromoters of 3-phosphoglycerate kinase or other glycolytic enzymes, thepromoters of phosphatase, for example, Pho5, α-mating systems of yeast,other sequences with different configurations or derivations known tocontrol expression of a prokaryotic or eukaryotic cell or their viruses,and various combinations thereof.

When a nucleic acid is disposed in a functional relationship with othernucleic acid sequence, it is operably linked to the nucleic acidsequence. An appropriate molecule (e.g., a transcription-activatingprotein) may be a gene and control sequence(s) that are linked in such amanner as to enable expression of the gene when it couple with thecontrol sequence(s). For example, DNA of a pre-sequence or a secretoryleader is operably linked to DNA of polypeptide when it is expressed asa pre-protein that participates in secretion of polypeptide; a promoteror enhancer is operably linked to a coding sequence when it affectstranscription of the coding sequence; an RBS is operably linked to acoding sequence when it affects transcription of the coding sequence; orthe RBS is operably linked to the coding sequence when it is disposed tofacilitate translation. In general, “operably linked” means that DNAsequences being linked are contiguous, and in case of the secretoryleader, contiguous and in a reading frame. However, an enhancer does nothave to contact a coding sequence. Linkage between sequences may beperformed by ligation in a convenient restriction enzyme site. However,when there is no restriction enzyme site, a synthetic oligonucleotideadaptor or linker may be used according to an ordinary method.

In the present invention, the term “expression vector” refers arecombinant carrier into which heterologous DNA fragments are inserted,wherein the DNA fragment is generally a double-strand DNA fragment.Here, heterologous DNA means hetero-type DNA that is not naturally foundin a host cell. Once the expression vector is incorporated with a hostcell, it can be replicated irrespective of host genomic DNA to generateseveral copies and their inserted (heterologous) DNAs.

As is known to one skilled in the art, in order to raise an expressionlevel of a transfected gene in a host cell, the corresponding gene mustbe operably linked to an expression control sequence that performstranscription and translation functions in a selected expression host.Preferably, the expression control sequence and the gene are included ina single expression vector comprising both a bacterial selectable markerand a replication origin. When an expression host is a eukaryotic cell,the expression vector must further include an expression marker usefulin the eukaryotic expression host.

In the present invention, various vectors including a plasmid vector, abacteriophage vector, a cosmid vector, and a yeast artificial chromosome(YAC) vector may be adopted as recombinant vectors. For the purpose ofthe present invention, a plasmid vector may be used as a recombinationvector. A typical plasmid vector that serves the purpose of theinvention includes (a) a replication origin that allows efficientreplication such that each host cell includes several hundred plasmidvectors, (b) an antibiotic-resistance gene that allows selection of ahost cell of which is transformed with a plasmid vector, and (c) arestriction enzyme cleavage site in which a foreign DNA fragment may beinserted. Even if there is no appropriate restriction enzyme cleavagesite, a vector may be easily ligated with foreign DNA by an ordinarymethod using a synthetic oligonucleotide adaptor or linker.

A recombinant vector according to the present invention may betransformed into an appropriate host cell using an ordinary method. Thehost cell may be bacteria, yeast, fungi and so on, but the presentinvention is not limited thereto. Preferably, the host cell may be aprokaryotic cell, for example, E. coli. Examples of appropriateprokaryotic host cells may include E. coli strain JM109, E. coli strainDH5a, E. coli strain JM101, E. coli K12 strain 294, E. coli strainW3110, E. coli strain X1776, E. coli XL-1Blue (Stratagene), E. coli B,etc. However, E. coli strains, such as FMB101, NM522, NM538, and NM539,and other prokaryotic species and genera may be also used. In additionto the above-described E. coli, Agrobacterium sp. strains such asAgrobacterium A4, bacilli such as Bacillus subtilis, otherenterobacteria such as Salmonella typhimurium or Serratia marcescens,and various Pseudomonas sp. strains may be used as host cells. However,the present invention is not limited to the above-described examples.

Also, the transformation of a eukaryotic cell may be easily accomplishedusing a calcium chloride method described in section 1.82, supra bySambrook et al. Alternatively, electroporation may be employed totransform theses cells (Neumann et al., EMBO J., 1:841 (1982)).

In order to prepare a plant containing a gene of a converting enzyme anda gene of a synthase according to the present invention, transfection ofthe plant may be performed by an ordinary method using Agrobacterium, avirus vector, etc. For example, an Agrobacterium sp. microbe istransformed with a recombinant vector containing the gene according tothe present invention, and the transformed Agrobacterium sp. microbe mayinfect tissue of a target plant, thereby obtaining a transfected plant.More specifically, preparation of a transfected plant may include (a)pre-culturing an explant of a target plant and co-culturing the explantwith transformed Agrobacterium to transfect the explant; (b) culturingthe transfected explant in a callus-inducting medium to obtain a callus;and (c) cutting and culturing the obtained callus in a shoot-inducingmedium to obtain a shoot.

In the present invention, the term “explant” refers to a tissue fragmentcut off from a plant and includes a cotyledon or a hypocotyl. Thecotyledon or hypocotyl may be used as an explant of a plant used for themethod of the present invention. The cotyledon, which is obtained bydisinfecting and cleaning a seed of a plant and germinating the seed ina Murashige and Skoog (MS) medium, may be preferably used.

In the present invention, target plants to be transfected may betobaccos, tomatoes, red peppers, beans, rice plants, corns, etc., butthe present invention is not limited thereto. Also, it is known to oneskilled in the art that even if a plant used for transformation issexually reproducible, it can be asexually reproduced by tissue culture,etc.

Advantageous Effects

As explained above, it was conventionally difficult to expresspropionyl-CoA transferase in E. coli, thereby precluding efficientsupply of lactyl-CoA. However, according to the present invention, amutant of propiony-CoA transferase from Clostridium propionicum isintroduced into recombinant E. coli to facilitate supply of lactyl-CoA,thereby preparing polylactate (PLA) and PLA copolymer with highefficiency.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of a intracellular synthesis pathway oflactate copolymer (poly(3HB-co-lactate)) using glucose, 3HB and lactate.

FIG. 2 is a schematic diagram of a process of preparing a recombinantexpression vector containing polyhydroxyalkanoate (PHA) synthase genefrom Pseudomonas sp. 6-19, and a mutant gene of propionyl-CoAtransferase from Clostridium propionicum, according to an example of thepresent invention.

MODE FOR INVENTION

Hereinafter, the present invention will be described in detail throughexamples. It is obvious to one skilled in the art that the examples isprovided only to explain the present invention in detail, the presentinvention is not limited to the examples

According to a previous invention of the present inventors disclosed inKorean Patent Application No 10-2006-0116234, in order to providelactyl-CoA that is a monomer required for synthesis of polylactate (PLA)and PLA copolymer, an operon-type constant expression system in whichpropionyl-CoA transferase from Clostridium propionicum (CP-PCT) andpolyhydroxyalkanoate (PHA) synthase are expressed together wasconstructed as will now be described in more detail.

Example 1-1 Cloning of a PHA Synthase Gene from Pseudomonas Sp. 6-19 andPreparation of Expression Vector

In order to separate PHA synthase ((phaC1_(Ps6-19)) gene fromPseudomonas sp. 6-19 (KCTC 11027BP) used in the present invention, thetotal DNA of Pseudomonas sp. 6-19 was extracted, primers having basesequences of SEQ ID NOs: 5 and 6 were prepared based on a sequence ofthe phaC1_(Ps6-19) gene (Ae-jin Song, Master's Thesis, Department ofChemical and Biomolecular Engineering, KAIST, 2004), and polymerasechain reaction (PCR) was performed to obtain the phaC1_(Ps6-19) gene.

SEQ ID NO: 5: 5′-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3′SEQ ID NO: 6: 5′-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3′

As a result of performing Agarose gel electrophoresis of the PCRproduct, a 1.7-kbp gene fragment corresponding to phaC1_(Ps6-19) genewas confirmed.

A DNA fragment containing a PHB-producing operon from Ralstonia eutrophaH16 was cut with BamHI/EcoRI from a pSYL105 vector (Lee et al., Biotech.Bioeng., 1994, 44:1337-1347), and inserted into a BamHI/EcoRIrecognition site of pBluescript II (Stratagene), thereby preparing apReCAB recombinant vector.

It is known that the pReCAB vector in which PHA synthase (phaC_(RE)) andmonomer-supplying enzymes (phaA_(RE) and phaB_(RE)) are constantlyexpressed by a PHB operon promoter, is also effectively operated in E.coli. (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347). The pReCABvector was cut with BstBI/SbfI to remove R. eutropha H16 PHA synthase(phaC_(RE)), and the phaC1_(Ps6-19) gene was inserted into a BstBI/SbfIrecognition site, thereby preparing a pPs619C1-ReAB recombinant vector.

In order to produce a phaC1_(Ps6-19) synthase gene fragment having onlya BstBI/SbfI recognition site on either end, an endogenous BstBI sitewas removed using site-directed mutagenesis (SDM) without change ofamino acids, and an overlapping PCR was performed using primers of SEQID NOs: 7 and 8, SEQ ID NOs:9 and 10, and SEQ ID NOs: 11 and 12 in orderto add the BstBI/SbfI recognition site.

SEQ ID NO: 7: 5′-ATG CCC GGA GCC GGT TCG AA-3′ SEQ ID NO: 8:5′-CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC-3′ SEQ ID NO: 9:5′-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3′ SEQ ID NO: 10:5′-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3′ SEQ ID NO: 11:5′-GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG-3′ SEQ ID NO: 12:5′-AAC GGG AGG GAA CCT GCA GG-3′

The base sequence of the phaC1_(Ps6-19) gene of the preparedpPs619C1-ReAB recombinant vector was confirmed by sequencing andrepresented by SEQ ID NO: 13, and the amino acid sequence coded therebywas represented by SEQ ID NO: 14.

The gene sequence similarity analysis results show that thephaC1_(Ps6-19) gene has a homology of 84.3% with phaC1 from Pseudomonassp. strain 61-3 (Matsusaki et al., J. Bacteriol., 180:6459, 1998) and anamino-acid sequence homology of 88.9%. Thus, it was confirmed that thetwo synthases were very similar enzymes. As a result, it was confirmedthat the phaC1_(Ps6-19) synthase obtained according to the invention wasa Type II PHA synthase.

Example 1-2 Preparation of a Substrate-Specific Mutant of a PHA Synthasefrom Pseudomonas sp. 6-19

Among various kinds of PHA synthases, a Type II PHA synthase is known asa medium-chain-length PHA (MCL-PHA) synthase for polymerizing asubstrate having relatively many carbon atoms, and the MCL-PHA synthaseis expected to be very applicable to production of a PLA copolymer.Although phaC1 synthase from Pseudomonas sp. 61-3, which has a highhomology with the phaC1_(Ps6-19) synthase obtained according to thepresent invention, is the Type II synthase, it was reported that thephaC1 synthase had a relatively wide range of substrate specificity(Matsusaki et al., J. Bacteriol., 180:6459, 1998), and results ofresearch in a mutant suitable for production of short-chain-length PHA(SCL-PHA) were reported (Takase et al., Biomacromolecules, 5:480, 2004).Based on the above, the present inventors found three amino-acid sitesaffecting SCL activation via amino-acid sequence arrangement analysis,and prepared mutants of phaC1_(Ps6-19) synthase by an SDM method usingprimers of SEQ ID NOs: 15 to 20 as shown in Table 1.

TABLE 1 Nucleic Amino acid acid Recombinant substi- substi- vectortution tution Primer pPs619C1200-ReAB AGC→ACC S325T SEQ ID NOs: 15/16CAG→ATG Q481M SEQ ID NOs: 17/18 pPs619C1300-ReAB GAA→GAT E130DSEQ ID NOs: 19/20 AGC→ACC S325T SEQ ID NOs: 15/16 CAG→ATG Q481MSEQ ID NOs: 17/18

SEQ ID NO: 15: 5′-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC-3′SEQ ID NO: 16: 5′-GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG-3′SEQ ID NO: 17: 5′-CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC-3′SEQ ID NO: 18: 5′-GCG GGT TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG-3′SEQ ID NO: 19: 5′-ATC AAC CTC ATG ACC GAT GCG ATG GCG CCG ACC-3′SEQ ID NO: 20: 5′-GGT CGG CGC CAT CGC ATC GGT CAT GAG GTT GAT-3′

Example 1-3 Preparation and Screening of a Library of a Mutant ofPropionyl-CoA Transferase from Clostridium propionicum

In the present example, in order to provide lactyl-CoA that is a monomerrequired for synthesis of PLA and PLA copolymer, propionyl-CoAtransferase from Clostridium propionicum (CP-PCT) was used and itssequence was represented by SEQ ID NO: 3. A fragment obtained byperforming PCR on chromosomal DNA of Clostridium propionicum usingprimers of SEQ ID NOs: 21 and 22 was used as CP-PCT. In this case, anNdeI site existing in wild-type CP-PCT was removed using SDM tofacilitate cloning.

SEQ ID NO: 21: 5′-GGA ATT CAT GAG AAA GGT TCC CAT TAT TAC CGC AGA TGA-3′SEQ ID NO: 22: 5′-GC TCT AGA TTA GGA CTT CAT TTC CTT CAG ACCCAT TAA GCC TTC TG-3′

Also, overlapping PCR was performed using a primer of SEQ ID NOs: 23 and24 in order to add a SbfI/NdeI recognition site.

SEQ ID NO: 23: 5′-agg cct gca ggc gga taa caa ttt cac aca gg-3′SEQ ID NO: 24: 5′-gcc cat atg tct aga tta gga ctt cat ttc c-3′

A pPs619C1300-ReAB vector containing phaC1_(Ps6-19)300, which is an SCLmutant of phaC1_(Ps6-19) synthase, was cut with SbfI/NdeI to remove amonomer-supplying enzyme (phaA_(RE) and phaB_(RE)) from R. eutropha H16,and the PCR-cloned CP-PCT gene was inserted into the SbfI/NdeIrecognition site, thereby preparing a pPs619C1300-CPPCT recombinantvector.

Example 2 Preparation and Screening of a Library of a Mutant ofPropionyl-CoA Transferase from Clostridium propionicum

As is known, when CP-PCT is highly expressed in E. coli, it causesserious metabolic disorder and exhibits toxicity. In general,recombinant E. coli were dead at the same time with addition of aninducer in an isopropyl-β-D-thio-galactoside (IPTG)-inducible proteinproduction system using a tac promoter or T7 promoter, which is widelyused to express recombinant proteins. For this reason, synthesis of PLAand PLA copolymer was performed using a constitutive expression systemwhich weakly but continuously expressed genes with growth ofmicroorganisms. In order to introduce random mutation into CP-PCT,pPs619C1300-CPPCT disclosed in Korean Patent Application No.10-2006-0116234 was used as a template, and error-prone PCR wasperformed using primers of SEQ ID NOs: 1 and 2 under conditions ofaddition of Mn²⁺ and the difference of concentration between dNTPs(refer to FIG. 2).

SEQ ID NO: 1: 5′-cgc cgg cag gcc tgc agg-3′ SEQ ID NO: 2:5′-ggc agg tca gcc cat atg tc-3′

Thereafter, in order to amplify a PCR fragment including randommutation, PCR was performed under common conditions using primers of SEQID NOs: 1 and 2. A pPs619C1300-CPPCT vector containingphaC1_(Ps6-19)300, which is an SCL mutant of phaC1_(Ps6-19) synthase,was cut with SbfI/NdeI to remove wild-type CP-PCT, and the amplifiedmutant PCR fragment was inserted into the SbfI/NdeI recognition site toproduce a ligation mixture. The ligation mixture was introduced into E.coli JM109, thereby preparing a CP-PCT library with a scale of about˜10^5 (refer to FIG. 2). The prepared CP-PCT library was grown for 3days in a polymer detection medium (a Luria Bertani (LB) agar, glucose20 g/L, 3HB 1 g/L, Nile red 0.5 n/ml) and screened to confirm whetherpolymer was generated, thereby primarily selecting ˜80 candidates. Thecandidates were grown for 4 days in a liquid medium (an LB agar, glucose20 g/L, 3HB 1 g/L, ampicillin 100 mg/L, 37° C.) under polymer generationconditions and analyzed using florescence activated cell sorting (FACS),thereby selecting final two samples. Also, a recombinant expressionvector containing the mutant was retrieved from E. coli and introducedinto E. coli JM109 again to confirm polymer yield. Thus, it wasconfirmed that the E. coli transformed with a recombinant vector havinga mutant of CP-PCT exhibited better performance than that havingwild-type CP-PCT.

Example 3 Preparation of a PLA Copolymer Using a Mutant of Propionyl-CoATransferase from Clostridium propionicum

In order to quantitatively analyze activation of mutants finallyselected in Example 2, E. coli JM109 transformed with the recombinantexpression vector containing the mutants as shown in FIG. 2 was culturedfor 4 days at a temperature of in a flask having an LB medium containingglucose (20 g/L) and 3HB (2 g/L). The cultured cell pellet was retrievedby centrifugation and dried for 24 hours in a dryer at a temperature of100. Thereafter, gas chromatography was performed to estimate thepolymer contents synthesized in cells as shown in Table 2.

TABLE 2 PLA Name of Strain content (w/w %) PHB content (w/w %)pPs619C1300-CPPCT/JM109 0.86%  5.85% CP-PCT mutant 512/JM109 2.19%12.82% CP-PCT mutant 522/JM109 7.49% 35.59%

As a result of the gas chromatographic analysis, it can be seen that therecombinant expression vector containing the mutant of CP-PCT preparedaccording to the present invention had about two to eight times thePLA-copolymer synthetic activity of the pPs619C1300-CPPCT vectorcontaining wild-type CP-PCT. This is because the mutant of the CP-PCTsupplies polymer synthesis monomers (i.e., lactyl-CoA and 3HB-CoA) moreefficiently than the wild-type CP-PCT.

In order to find out the mutant positions of the prepared CP-PCTmutants, gene sequencing was performed with respect to the CP-PCTmutants, and the results were shown in Table 3.

TABLE 3 Recombinant vector Nucleic acid substitution CP-PCT mutant 512A1200G CP-PCT mutant 522 T78C, T669C, A1125G, T1158C

As a result of the gene sequencing of the CP-PCT mutants, onenucleic-acid substitution occurred in the mutant 512, while fournucleic-acid substitutions occurred in the mutant 522. However, it wasconfirmed that all the nucleic-acid substitutions were silent mutationsthat cause no amino-acid substitution. That is, it was assumed that animprovement in monomer-supplying capability of the CP-PCT mutantsprepared according to the present invention resulted not from anincrease in activity due to amino-acid substitution of an enzyme butfrom a variation in the enzyme expression in E. coli. Thus, the codonusages of gene sequences of the wild-type CP-PCT and CP-PCT mutantsprepared according to the present invention in typical E. coli wereanalyzed as shown in Table 4.

TABLE 4 78 669 1125 1158 1200 Wild-type CP-PCT GGT (Gly) GGT (Gly) AAA(Lys) CGT (Arg) ACA (Thr) 24.7 24.7 33.6 20.9 7.1 CP-PCT mutant 512 GGT(Gly) GGT (Gly) AAA (Lys) CGT (Arg) ACG (Thr) 24.7 24.7 33.6 20.9 14.4 CP-PCT mutant 522 GGC (Gly) GGC (Gly) AAG (Lys) CGC (Arg) ACA (Thr) 29.629.6 10.3 22.0 7.1

As shown in Table 4, all the nucleic-acid substitutions except A1125Gexisting in the mutant 522 were advantageous for the codon usage of E.coli. That is, due to the nucleic-acid substitution being advantageousfor the codon usage of the E. coli, the CP-PCT mutants preparedaccording to the present invention increased the expression of anactivated enzyme and thus exhibited higher monomer-supplyingcapabilities required for production of PLA copolymers.

Example 4 Preparation of a PLA Copolymer Using a Mutant of Propionyl-CoATransferase from Clostridium propionicum

Random mutagenesis was performed on the mutants (512 and 522) finallyselected in Example 2, in the same manner as described in Example 2,thereby obtaining the following CP-PCT mutants 531-537 and 540.

In order to quantitatively analyze the CP-PCT mutants, E. coli JM109transformed with the recombinant expression vector containing the CP-PCTmutants 531-537 and 540 was cultured for 4 days at a temperature of 30°C. in a flask having a P-rich methyl red (MR) medium containing glucose(20 g/L) and 3HB (2 g/L). The cultured cell pellet was retrieved bycentrifugation and dried for 24 hours in a dryer maintained at atemperature of 100° C. Thereafter, gas chromatography was performed toestimate the contents of polymers synthesized in cells. The results ofgene sequencing with respect to the CP-PCT mutants performed to find outthe mutation positions of the CP-PCT mutants are shown in Table 5, andthe contents of the polymers synthesized in the cells are shown in Table6.

In the above-described experiment, P-rich MR was formed of 22 g ofKH₂PO₄; 3 g of (NH₄)₂HPO₄; 0.8 g of Citrate; 0.7 g of MgSO₄.7H₂O; and 5mL of a trace metal solution per liter, and the trace metal solution wasformed of 10 g of FeSO₄.7H₂O; 2.25 g of ZnSO₄.7H₂O; 1 g of CuSO₄.5H₂O;0.5 g of MnSO₄.5H₂O; 2 g of CaCl₂.2H₂O; 0.23 g of Na₂B₄O₇.7H₂O; 0.1 g of(NH₄)₆Mo₇O₂₄; and 10 mL of HCl 35% per liter.

TABLE 5 Mutations Silent Mutations CpPct512 A1200G CpPct522 T78C, T669C,A1125G, T1158C CpPct531 Gly335Asp A1200G CpPct532 Ala243Thr A1200GCpPct533 Asp65Gly T669C, A1125G, T1158C CpPct534 Asp257Asn A1200GCpPct535 Asp65Asn T669C, A1125G, T1158C CpPct537 Thr199Ile T669C,A1125G, T1158C CpPct540 Val193Ala T78C, T669C, A1125G, T1158C

TABLE 6 Name Polymer content (%) Lac mol % 3HB mol % PCT (wild control)24.9 38.0 62.0 531 26.5 56.5 43.5 532 23.5 54.5 45.5 533 25.2 63.8 36.2534 23.9 58.0 42.0 535 30.7 59.2 40.8 537 33.3 52.4 47.6 540 23.7 21.378.7

As shown in Table 6, the CP-PCT mutants prepared according to thepresent invention remarkably increased lactate mole % compared towild-type CP-PCT. Also, it was confirmed that the recombinant expressionvector containing the mutants 531, 533, 535, 537, and 540 exhibitedbetter PLA-copolymer synthetic activity than the vector containingwild-type CP-PCT.

While the invention has been shown and described with reference tocertain examples thereof, it will be understood by those skilled in theart that various changes in form and details may be made therein withoutdeparting from the spirit and scope of the invention as defined by theappended claims.

The invention claimed is:
 1. An isolated mutant of propionyl-CoAtransferase supplying lactyl-CoA, which has a gene sequence of SEQ IDNO: 3, in which T669C, A1125G and T1158C are mutated, wherein thepropionyl-CoA transferase comprises a) a gene sequence of SEQ ID NO: 3,in which T669C, A1125G and T1158C are mutated and one mutation of thenucleic acid is further introduced to cause mutation of Asp65Gly; b) agene sequence of SEQ ID NO: 3, in which T669C, A1125G and T1158C aremutated and one mutation of the nucleic acid is further introduced tocause mutation of Asp65Asn; c) a gene sequence of SEQ ID NO: 3, in whichT669C, A1125G and T1158C are mutated and one mutation of the nucleicacid is further introduced to cause mutation of Thr199Ile; or d) a genesequence of SEQ ID NO: 3, in which T78C, T669C, A1125G and T1158C aremutated and one mutation of the nucleic acid is further introduced tocause mutation of Val193Ala.